Fusion expression,detection and application of HHV-8 antigen / 重庆医学

Objective To construct the prokaryotic expression plasmid of HHV-8 fusion antigen for diagnosis of HHV-8 infec-tion .Methods The combined fragment ORF59 ,ORF65 and K8 .1 by fusion PCR was integrated into pQE-80L and transfected into E .coli DH5α.Fusion protein was induced to express by IPTG .SDS-PAGE and Western blot were employed to detect the fusion protein .Fusion protein was used to detect serum of blood donors .Results The combined plasmid pQE-80L-ORF59-ORF65-K8 .1 was constructed successfully after verifying by restriction enzyme digestion and sequencing .The fusion protein was about 24 KD and could be specific combined with HHV-8 positive serum .The fusion protein had the same result to detect HHV-8 with the HHV-8 ELISA kit .Conclusion Fusion protein we construct can be used as diagnosis antigen to detect HHV-8 of blood donors and common people .

Expression of Low Molecular Weight Keratin (K8/18) in Fetal Skin Development / 대한피부과학회지

BACKGROUND: The epidermis and adnexal epithelium might express different types of keratin (K) during fetal development. OBJECTIVE: The objective is to observe the distribution of K8/18 in the skin of fetuses and to find out the distinction of expressions of K8/18 during fetal development. METHODS: Immunohistochemical analysis was applied to the skin of the scalp and sole of 42 fetuses ranging from 10 to 39 weeks of gestation. Immunohistochemical staining with monoclonal antibodies with CAM5.2 using LSAB kit against K8/18 was conducted. RESULTS: In the skin of the scalp, K8/18 was expressed in the periderm and basal layer of epidermis from the 10th week to the 31st week of fetal gestation. K8/18 was expressed in the hair germ, bulge and basal cells of fetal the infundibulum and sebaceous glands. Root sheath cells were weakly positive but matrix cells were negative. The expression of K8/18 was negative in the basal layer of the sole. Merkel cells, which are located in the basal layer and upper dermis, were positive from the 12th week of gestation. Terminal eccrine ducts and acinar cells were positive after the 20th week of gestation. CONCLUSION: K8/18 in the skin of the scalp and sole of fetuses were expressed in different ways. The expression of K8/18 in the basal cells of the sole were negative while basal cells of the epidermis of the scalp were positive transiently from the 12th to the 31st week of gestation. Early hair germ cells and bulge cells were expressed strongly in hair follicles. Terminal eccrine ducts and acini were expressed strongly in the eccrine gland. Merkel cells located in the basal layer and papillary dermis also express K8/18.

Effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the Expression of Keratin in HaCaT Cells / 대한해부학회지

In human skin, specific keratin markers reflect on normal differentiation and pathologic conditions. This experiment focused on the expressional pattern of keratin 10 (K10: normal differentiation marker), and keratin 8 & 13 (K8 & K13: pathologic differentiation marker) together with their cellular localization after treating HaCaT cells with 12-Otetradecanoylphorbol 13-acetate (TPA). The cells were treated with TPA at 0, 0.1, 1 microgram/ml for 2 hours or 6 hours. Morphologic studies revealed that TPA treatment changed the shape of cells into the fibroblast-like cells with highly folded nuclear membrane and reduced number of the desmosome. The results of indirect immunofluorescent staining and Northern blotting analysis showed that TPA considerably down-regulated the expression of K10, while markedly up-regulating the expression of K8 and K13 both at protein and mRNA levels. Furthermore, by simultaneous staining for keratins and DNA content in flow cytometry, it was found that TPA increased the expression of K8 and K13 dramatically at the S-G2-M phase of the cells. In conclusion, these changes induced by TPA in HaCaT cells may indicate a close relationship between the morphologic change and the altered expression of keratin subfamilies. It also suggests that TPA known as a tumor promotor may directly induce the potentially malignant cells even without the support of tumor initiator.

Change in Expression of Keratin and Proto-oncogene Induced by Beta-propiolactone in HaCaT Cell / 대한해부학회지

To investigate the relationship between the morphologic changes and the expression of keratin and proto-oncogene induced by Beta-propiolactone (BPL), we assessed on the expression of keratins (K8, K10, K13) and proto-oncogenes (c-fos, c-jun, c-myc) in human HaCaT cell line. The cells were treated with 0, 0.1, 1 microgram/ml BPL for 2 or 6 hours. Morphologic studies revealed that BPL changed the cells into fibrocyte-shaped, caused highly lobulated nuclei and reduced desmosomes in their number. Findings of immunofluorescence and Northern blotting indicated that BPL consistently decrease expression of K10 representing a normal differentiation marker of keratinocytes, while increasing expression of K8 and K13 associated with a pathologic differentiation. This reagent also up-regulated expression of c-fos and c-jun, and down-regulated expression of c-myc. Together with staining for each keratin or proto-oncogene and DNA content in flow cytometry, BPL increased K8 expression dramatically at S-G2-M phase. The induction of c-Fos at S-G2-M phase appeared within 2 hours, and c-Jun gradually occurred. However, c-Myc was inhibited regardless of phases of cell cycle. In conclusion, these changes caused by BPL demonstrate a close relationship between the morphologic evidence and the altered expression of each keratin and proto-oncogene.